Brain aromatase is widely distributed in the vertebrates, from fish to mammals, and plays important roles in functional reproductive behavior through production of estrogen as a neurosteroid. It is expressed only in the nerve cells of specific brain regions with a transient peak in the neonatal period when sexual behavior becomes organized, and therefore provides a good model system to study regulatory mechanism of cell-specific, brain region-specific, and developmental stage-specific expression. To elucidate spatiotemporal regulation of brain aromatase, we prepared transgenic mice carrying a reporter gene under the promoter of brain-specific exon 1f of the mouse aromatase gene. The reporter transgene carrying a 6.5kb upstream region of the brain-specific promoter accurately reproduced the spatiotemporal expression patterns of aromatase in mouse brain, whereas transgenes carrying smaller fragments of the promoter showed ambiguous or inconsistent expression patterns. The binding sites of Aro-AI, Aro-AII, and Aro-B for nuclear factors were also identified in the proximal region of the exon 1f brain-specific promoter. Introduction of a mutation into the Aro-AII site in the reporter transgene carrying −6.5kb promoter region of exon 1f caused complete alteration of the spatiotemporal expression pattern of the reporter gene in the transgenic mice. These results indicate that the −6.5kb promoter region of exon 1f is the minimal essential element for brain-specific regulation, with both proximal and distal promoter regions required for accurate spatiotemporal expression of aromatase in the mouse brain.